Procalcitonin (PCT) plays a significant role in screening for bacterial infection (BI), which is one of the world's leading health issues. This work presents a simple, efficient, and convenient glass fiber paper (GFP) substrate-based innovative biosensor application for BI-specific PCT biomarker detection. To enhance the conductivity of the GFP substrate, a self-accumulating single layer of AuNP is used on the GFP surface. This monolayer of AuNP in GFP provides a platform for antibody immobilization and shows high electrochemical conductivity. Further, the AuNP/GFP electrode was modified with antibodies specific to PCT, followed by the addition of bovine serum albumin (BSA) to block unspecific binding sites for PCT detection. The PCT/BSA/ab/AuNP/GFP bioelectrode has been successfully used to determine PCT in a serum sample with amended stability. The structural and morphological features were characterized using scanning electron microscopy, transmission electron spectroscopy, Fourier transforms infrared, ultraviolet-visible spectroscopy, and X-ray diffraction. Using an electrochemical impedance response study, the developed PCT/BSA/ab/AuNP/GFP bioelectrode showed the lowest detection limit of 100 pg ml(-1) concerning PCT detection in the linear range of 10(7)-10(2) pg ml(-1). Electrochemical response studies show that the developed BSA/ab/AuNP/GFP bioelectrode has a lifespan of 30 days and can be used to assess PCT in the 10(7)-10(2)pg ml(-1) range. Moreover, this evaluated sensor offers many merits, including a lower cost, small sample volume, single-use, and rapid analysis over conventional methods, making this platform an alternative option for PCT screening. This designed bioelectrode also shows adequate reproducibility.